The following discussion of the background of the invention is merely provided to aid the reader in understanding the invention and is not admitted to describe or constitute prior art to the present invention.
The explosion of recent knowledge in basic genetics has spawned numerous clinical follow-up studies that have confirmed an unequivocal association between the presence of specific prevalent genetic alterations and susceptibility to some very common human diseases. In addition, the Human Genome Project's sequencing efforts will contribute yet more candidate disease genes that will require both research-based genetic association studies (to confirm suspected disease links) and, if positive, the translation of these disease-genotype associations to routine diagnostic clinical practice. Given this expanding repertoire of confirmed and reputed disease genes (many for common diseases), the demand for rapid, sensitive, specific, inexpensive assays for their clinical- and/or research-based detection is growing quickly.
As a consequence, clinical genetic testing laboratories, once accustomed to manual, low-volume, high-labor tests on patients with rare, untreatable classic “genetic” diseases, will soon need to develop better high-throughput and semi-automated methods. In the fast-approaching molecular medicine era, these new genotyping methods will be utilized not only for diagnosing symptomatic patients but perhaps, more importantly, for presymptomatically identifying individuals at risk for common, treatable diseases for whom effective preventative interventions may be available.
Oligonucleotide hybridization is a method commonly used in the field of molecular biology for the treatment and diagnosis of disease, as well as the identification, quantitation, and isolation of nucleic acids. Accordingly, it is important to identify methods to increase the specificity and affinity of oligonucleotides for their targets. In this way, diagnostics which provide efficient and precise answers can be made. Various methods for increasing the specificity of oligonucleotides are known in the art, including increasing the length, choosing oligonucleotides that are not likely to cross-hybridize or bind non-specifically and designing oligonucleotides that have a high annealing temperature. (See e.g., Bergstrom et al., J. Am. Chem. Soc. 117:1201-1209, 1995; Nicols et al., Nature 369:492-493, 1994; Loakes, Nucl. Acids Res. 22:4039-4043, 1994; Brown, Nucl. Acids Res. 20:5149-5152, 1992).
U.S. Pat. No. 5,780,223 discloses “an improved nucleic acid hybridization process . . . which employs a modified oligonucleotide”, wherein “the modified probe contains at least one artificial mismatch”. “Suitable natural or non-natural artificial mismatches are, therefore, preferably universal mismatches.” U.S. Pat. No. 5,780,223 indicates that when creating more than one artificial mismatch, “a spacing of 10 nucleotides between artificial mismatches is desired”. In addition, U.S. Pat. No. 5,780,223 indicates that “artificial mismatch positions account for no more than about 20% of the total number of positions in a probe”.
As another example, U.S. Pat. No. 6,361,940 states that the incorporation of a “specificity spacer” that “cannot enter into hydrogen bonding with a base positioned opposite itself in a hybridized complementary base sequence” is capable of “increasing the specificity of a probe nucleic acid for a target nucleic acid”. U.S. Pat. No. 6,361,940 indicates that “no two specificity spacers should be adjacent to one another”, preferably “separated by 4-14 nucleotides having a wild-type sequence”.